The overall goal of this new AREA (R-15) research proposal is to develop, validate, and utilize a zebrafish-based model system to elucidate the cellular and molecular details of gastrointestinal (Gl) function with an emphasis on motility. A new model system exploring Gl physiology is needed because current therapies for Gl motility disorders are lacking. A zebrafish-based model system will provide several important experimental advantages, such as the direct visualization of Gl motility in optically transparent larvae and the opportunity to utilize a forward genetic approach to identify novel molecular components determining Gl motility. The Gl tract appears functional by 5 days post-fertilization when larvae swallow food, exhibit peristalsis, and absorb nutrients. In Specific Aim 1 the cellular components that regulate Gl motility in mammals, interstitial cells of Cajal (ICC), will be characterized in larvae and adults, using immunohistochemical procedures. Expression of the kit gene specifically identifies ICC, and kit expression will identify putative ICC in the zebrafish. Development of ICC, or pacemaker cells, is expected to coincide with development of spontaneous contractions, similar to other vertebrates. The anatomical position of ICC, intermediary between enteric neurons and Gl smooth muscles, indicates the functional role of integration and coordination of motility in humans. Anatomical features of putative ICC cells and networks will be determined. In Specific Aim 2 the functional significance of kit-positive cells will be determined using wild type and null kit mutant zebrafish. Normal motility patterns, including the frequency of spontaneous contractions and the time for food to clear the Gl tract, will be determined. The dependence of motility on the pacemaker cell will be examined using zebrafish with inactivating kit mutations, which lesion ICC in other vertebrate models. A zebrafish based model system has the potential for the identification of novel genes regulating Gl motility, which may benefit treatment for disorganized motility.